Localization-based super-resolution microscopy with an sCMOS camera

Abstract

In the community of localization-based super-resolution microscopy (or called localization microscopy), it is generally believed that the emission of single molecules is so weak that an EMCCD (electron multiplying charge coupled device) camera is necessary to be used as the detector by eliminating read noise. Here we evaluate the possibility of a new kind of low light detector, scientific complementary metal-oxide-semiconductor (sCMOS) camera in localization microscopy. We demonstrate experimentally that sCMOS is capable of imaging actin bundles with FWHM diameter of 37 nm, evidencing the capability of sCMOS in localization microscopy. We further characterize the noise performance of sCMOS and find out that, with the use of a bright fluorescence probe such as d2EosFP, localization microscopy imaging is now working in the shot noise limited region.

Advanced methods of microscope control using μManager software

“…The fastest sCMOS cameras can produce 100 frames per second of 2560×2160 pixels each, or 1.1 GB/s. High speed sCMOS cameras have proven useful for imaging three-dimensional evolving specimens [5] and advanced applications such as super-resolution microscopy [6]. As of release 1.4.15, μManager is optimized to continuously acquire images from these cameras at full speed, and stream the images to disk or store them in RAM.…”

Super-resolution fluorescence polarization microscopy

“…The modulation could be in a structured manner, such as stimulated emission depletion (STED) 54,55 or (saturated) structured illumination microscopy (SIM), [56][57][58] or in a stochastic manner, such as (f) photo-activated localization microscopy (PALM)/ stochastic optical reconstruction microscopy (STORM). [59][60][61][62] Though a large variety of super resolution techniques have been developed for°uo-rescence intensity imaging, super resolution FPM is just starting. To achieve super resolution with dipole orientation measurement, one simple thought is to combine FPM with existing super resolution techniques, such as STED, SIM, PALM/STORM, etc.…”

Assessing low-light cameras with photon transfer curve method

“…17,18 Basing on our previous works, 13,21 here we present brie°y such procedures to help readers understand the PTC method.…”

“…12 This new type of low-light camera has attracted great attention ever since its announcement. The capabilities of sCMOS in various applications, including super-resolution localization microscopy, [13][14][15] have been tested and reported in the past several years.…”